RNA interference refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering RNAs (siRNA). After the discovery of the phenomenon in plants in the early 1990s, Andy Fire and Craig Mello demonstrated that double-stranded RNA (dsRNA) specifically and selectively inhibited gene expression in an extremely efficient manner in Caenorhabditis elegans (Fire et al., 1998, Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans. Nature, 391:806). The sequence of the first strand (sense RNA) coincided with that of the corresponding region of the target messenger RNA (mRNA). The second strand (antisense RNA) was complementary to the mRNA. The resulting dsRNA turned out to be several orders of magnitude more efficient than the corresponding single-stranded RNA molecules (in particular, antisense RNA).
The process of RNAi begins when the enzyme, DICER, encounters dsRNA and chops it into pieces called small-interfering RNAs (siRNA). This protein belongs to the RNase III nuclease family. A complex of proteins gathers up these RNA remains and uses their code as a guide to search out and destroy any RNAs in the cell with a matching sequence, such as target mRNA (see Bosher & Labouesse, 2000, RNA interference: genetic wand and genetic watchdog. Nat Cell Biol, 2000, 2 (2):E31, and Akashi et al., 2001, Suppression of gene expression by RNA interference in cultured plant cells. Antisense Nucleic Acid Drug Dev, 11 (6):359).
In attempting to utilize RNAi for gene knockdown, it was recognized that mammalian cells have developed various protective mechanisms against viral infections that could impede the use of this approach Indeed, the presence of extremely low levels of viral dsRNA triggers an interferon response, resulting in a global non-specific suppression of translation, which in turn triggers apoptosis (Williams, 1997, Role of the double-stranded RNA-activated protein kinase (PKR) in cell regulation. Biochem Soc Trans, 25 (2):509; Gil & Esteban, 2000, Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): mechanism of action. Apoptosis, 5 (2):107-14).
In 2000 dsRNA was reported to specifically inhibit 3 genes in the mouse oocyte and early embryo. Translational arrest, and thus a PKR response, was not observed as the embryos continued to develop (Wianny & Zernicka-Goetz, 2000, Specific interference with gene function by double-stranded RNA in early mouse development. Nat Cell Biol, 2 (2):70). Research at Ribopharma AG (Kulmbach, Germany) demonstrated the functionality of RNAi in mammalian cells, using short (20-24 base pairs) dsRNA to switch off genes in human cells without initiating the acute-phase response. Similar experiments carried out by other research groups confirmed these results. (Elbashir et al., 2001, RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev, 15 (2):188; Caplen et al., 2001, Specific inhibition of gene expression by small double stranded RNAs in invertebrate and vertebrate systems. Proc. Natl. Acad. Sci. USA, 98: 9742). Tested in a variety of normal and cancer human and mouse cell lines, it was determined that short hairpin RNAs (shRNA) can silence genes as efficiently as their siRNA counterparts (Paddison et al, 2002, Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev, 16 (8):948). Recently, another group of small RNAs (21-25 base pairs) was shown to mediate downregulation of gene expression. These RNAs, small temporally regulated RNAs (stRNA), regulate timing of gene expression during development in Caenorhabditis elegans (for review see Banerjee & Slack, Control of developmental timing by small temporal RNAs: a paradigm for RNA-mediated regulation of gene expression. Bioessays, 2002, 24 (2):119-29 and Grosshans & Slack, 2002, Micro-RNAs: small is plentiful. J Cell Biol, 156 (1):17).
Scientists have used RNAi in several systems, including Caenorhabditis elegans, Drosophila, trypanosomes, and other invertebrates. Several groups have recently presented the specific suppression of protein biosynthesis in different mammalian cell lines (specifically in HeLa cells) demonstrating that RNAi is a broadly applicable method for gene silencing in vitro. Based on these results, RNAi has rapidly become a well recognized tool for validating (identifying and assigning) gene function. RNAi employing short dsRNA oligonucleotides will yield an understanding of the function of genes that are only partially sequenced.
The transient receptor potential vanilloid-1 (TRPV1), also called Vanilloid receptor 1 (VR-1), is a capsaicin-responsive ligand-gated cation channel, that was first discovered in 1997 (Caterina et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature. 1997 Oct. 23; 389 (6653):816-24). TRPV1 is mainly expressed on sensory neurons and serves as a molecular detector for heat, capsaicin, protons, and endovanilloids (Caterina M J & Julius D. The vanilloid receptor: a molecular gateway to the pain pathway. Annu Rev Neurosci., 2001; 24:487-517; Montell et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev., 2002, 16 (8):948-58; Baumann T K & Martenson M E. Extracellular protons both increase the activity and reduce the conductance of capsaicin-gated channels. J Neurosci. 2000; 20:RC80).
When TRPV1 is activated by agonists such as capsaicin and other factors such as heat, acidosis, lipoxygenase products or anandamide, calcium enters the cell and pain signals are initiated. Activation of the channel induces neuropeptide release from central and peripheral sensory nerve terminals, resulting in the sensation of pain, neurogenic inflammation, and sometimes, in smooth muscle contraction and cough. Recent evidence suggests a role of TRPV1 in pain, cough, asthma and urinary incontinence (Jia et al., TRPV1 receptor: a target for the treatment of pain, cough, airway disease and urinary incontinence. Drug News Perspect. 2005 April; 18 (3):165-71).
Due to the fact that both the sensitivity and the density of expression of TRPV1 are enhanced during inflammatory conditions (Di Marzo et al., Endovanilloid signaling in pain. Curr Opin Neurobiol. 2002 August; 12 (4):372-9), downregulation of TRPV1 expression and/or activity is a promising therapeutic strategy for novel analgesic drugs. As a matter of fact, intraperitoneal administration of selective TRPV1 blockers into mice proved to attenuate chemical and thermal nociception and hyperalgesia (Garcia-Martinez et al., Attenuation of thermal nociception and hyperalgesia by VR1 blockers. Proc Natl Acad Sci USA. 2002 Feb. 19; 99 (4):2374-9).
TRPV1 channel function is upregulated by several endogenous mediators present in inflammatory conditions, which decrease the threshold for activation of the channel. Thus, it has recently been demonstrated that acute pain-related behaviour evoked by elevated ionic strength is abolished in TRPV1-null mice and inhibited by iodoresiniferatoxin, a potent TRPV1 antagonist (Ahern et al., Extracellular cations sensitize and gate capsaicin receptor TRPV1 modulating pain signaling. J Neurosci. 2005 May 25; 25 (21):5109-16). Further, Prostaglandin E2 (PGE2) and Prostaglandin I2 (PGI2) have proven to increase or sensitize TRPV1 responses through their respective receptors EP1 or IP (Moriyama et al., Sensitization of TRPV1 by EP1 and IP reveals peripheral nociceptive mechanism of prostaglandins. Mol Pain. 2005 Jan 17; 1 (1):3), suggesting for the first time that sensitisation of TRPV1 activity through EP1 or IP activation might be one important mechanism underlying the peripheral nociceptive actions of PGE2 or PGI2. WO 2004/042046 shows that siRNA targeted against VR1 can be used in the treatment of chronic pain, sensitivity disfunctions linked to the VR1 receptor and VR associated inflammation, tumours urinary incontinence and pruritus.
Polymodal nociceptors are the most abundant nociceptor type found in the cornea. There exists pharmacological evidence that these receptor fibers express the TRPV1 receptor because they respond to capsaicin, heat and acid. Moreover, high doses of capsaicin inactivate the activation of corneal polymodal nociceptors to heat and acid whereas mechanical responsiveness remains unaffected. This suggests that TRPV1 receptors present in corneal polymodal nerve endings were selectively inactivated. Therefore, it is likely that an important part of the acute nociceptive response to corneal injury and the sustained pain sensations that accompany inflammatory and irritative processes in this tissue are mediated by TRPV1 activation.
Recent evidence also demonstrates that both insulin and IGF-I enhance TRPV1-mediated membrane currents in heterologous expression systems and cultured dorsal root ganglion neurons (Van Buren et al., Sensitization and translocation of TRPV1 by insulin and IGF-I. Mol Pain. 2005 Apr. 27; 1 (1):17). Enhancement of membrane currents results from both increased sensitivity of the receptor and translocation of TRPV1 from cytosol to plasma membrane. An increase of IGF-1 has been found in the serum (Merimee et al., Insulin-like growth factors. Studies in diabetics with and without retinopathy. N. Engl. J. Med., 1983; 309:527-530; Grant et al., Insulin-like growth factors in vitreous. Studies in control and diabetic subjects with neovascularization. Diabetes, 1986; 35:416-420) and the vitreous body and intraocular fluid (Grant et al., 1986; Inokuchi et al., Vitreous levels of insulin-like growth factor-I in patients with proliferative diabetic retinopathy. Curr. Eye Res., 2001; 23:368-371) of patients with diabetic retinopathy. Further, vitreous IGF-I levels correlate with the presence and severity of ischemia-associated diabetic retinal neovascularization (Meyer-Schwickerath et al., Vitreous levels of the insulin-like growth factors I and II, and the insulin-like growth factor binding proteins 2 and 3, increase in neovascular eye disease. Studies in nondiabetic and diabetic subjects. J Clin Invest., 1993; 92 (6):2620-5). However, the source of increased ocular IGF-1 in retinopathy is controversial, and the relative contribution of either endogenous IGF-1 or serum IGF-1 is unknown (Ruberte et al., Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease. J Clin Invest. 2004 April; 113 (8):1149-57). Modulation of TRPV1 levels could aid in the control of diabetic retinopathy mediated by IGF-I.
Although originally described on sensory neurons, TRPV1 has now been detected in several human skin cell populations and epithelial compartments of the human hair follicle (HF), mainly the outer root sheath (ORS) and hair matrix (Bodo et al., A hot new twist to hair biology: involvement of vanilloid receptor-1 (VR1/TRPV1) signaling in human hair growth control. Am J Pathol. 2005 April; 166 (4):985-98). Stimulation of TRPV1 in organ culture and cultured human ORS keratinocytes inhibits proliferation, induces apoptosis, elevates intracellular calcium concentration, up-regulates known endogenous hair growth inhibitors, and down-regulates known hair growth promoters, thus supporting TRPV1 as a significant novel player in human hair growth control (Bodo et al., 2005).
The above-mentioned evidence points to inhibition of TRPV1 as an efficient treatment for eye conditions that mediate with an excess of expression and/or activity of TRPV1, such as discomfort and altered sensitivity of the cornea following refractive surgery, use of contact lenses and dry eyes. The functional relationship between TRPV1 and IGF-I highlights the importance of downregulation of TRPV1 for the treatment of diabetic retinopathy mediated by high levels of IGF-I. The role played by TRPV1 in human hair follicle growth and keratinocytes targets TRPV1 as a good candidate to be inhibited for the treatment of hair follicle and skin abnormal conditions such as alopecia.